1. Field of the Invention
The invention relates to the field of electrospray ionization mass spectroscopy (ESI-) and, more particularly, to the discovered use of a tagging reagent to associate with neutral compounds to provide tagged compounds suitable for detection and analysis using mass spectroscopy analysis. The present invention also relates to novel compounds discovered using such ESI-methods, including fulleroids.
2. Description of the Background Art
The use of electrospray ionization mass spectrometry (ESI-MS) for analysis of chemical compounds is under investigation (1).
ESI-MS is a developing new technique; and J. Organometallics, in press). Electrospray (J. B. Fenn et al., Science 246:64 (1989); Mass Spectrometry Rev., 9:37 (1990); and R. D. Smith et al. Anal. Chem., 62:882 (1990)) in a technique for directly spraying a solution of ions into amass spectrometer. The technique is so gentle that only molecular ions, characteristic of the molecular weights of the compounds of interest, are seen. The technique of electrospray is therefore a method for "weighing" molecules in dilute solution. The structures of the detected ions can often be deduced from the electrospray spectrum. While electrospray has recently revolutionized the mass measurement of biological molecules, applications of small organic molecules are rare.
For detection, ESI-MS requires the presence of cationic groups or anionic groups. Neutral compounds have been poor candidates for analysis by ESI-MS for several reasons. For example, neutral small compounds often are volatile and may readily be determined using GC-MS. Even so, neutral compounds are often derivatized to make them more thermally stable and volatile. Neutral compounds also are not directly detectable by ESI-MS since they are not charged. However, since many chemical reactions take place in solution, there would be significant advantages to the use of ESI-MS for analyzing neutral compounds, since this technique allows the analysis of solution chemistry.
Maquin et al, Rapid Commun. Mass. Spectrom. (England) 5(6):299-392 (June 1991) discloses that the molecular weights of recombinant protein interleukin-2 and interferon gamma were determined by ionization (ESI) mass spectroscopy on the charged molecules. The interleukin-2 was found to have an average experimental mass of 15,549.4 u, and a mass observed for the interferon gamma was 16,908.4 u.
DeSerrano et al, Arch. Biochem. Biophys. (United States) 294(1):282-290 (April 1992) discloses molecular mass determination of a recombinant domain from tissue-type plasminogen activator as 9621.9.+-.4.0.
Rumb et al, FEBS Lett (Netherlands) 296(2):153-157 (Jan. 20, 1992) discloses the characterization of mutant Asp-52 and lysozyme with GSE NAC4 and GLC NAC6.
Romk et al, Biochemistry 30(39):9435-9442 (Oct. 1, 1991) discloses structural characterization of rusticyanin isolated from Thiobacillus ferrooxidans using, inter alia, triple-quadrapole mass spectrometry techniques.
Henion, U.S. Pat. No. 4,935,624, discloses a thermo-assisted electrospray interface used in series between a liquid chromatograph and a mass analyzer to give increased sensitivity for detection of components in a liquid stream at high flow rates up to 2 ml per minute. This device provides a combination of thermo-energy and electric field potential to disperse the liquid into a fine mist which is then directed at atmospheric pressure into the ionization chamber of a mass spectrometer. The invention was tested on a series of compounds including disulphonated acyl dyes, phenolic compounds, carboxylic acids, drug compounds, nucleotides and glycopolymers.
Hiraoka et al, Rapid. Comm. Mass Spect. 6:25-256 (1992) attempted detections of fullerene ions C.sub.60.sup.- and C.sub.70.sup.- using ESI-MS. Attempts to detect cations of C.sub.60 K.sup.+ or C.sub.70 K.sup.+ using the most suitable 50/50 (v/v) methanol+benzene solvent failed, even though K.sup.+ (H.sub.2 O), K.sup.+ (CH.sub.3 OH) and K.sup.+ (C.sub.6 H.sub.6) produced strong signals. In alternative positively charged fullerenes by electron transfer between naphthacene and 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) in CH.sub.2 Cl.sub.2 +CH.sub.3 OH (50/50, v/v), the ionization energies of the resulting radical cation of naphthacene and of C.sub.60 would theoretically be of the same order. However, such a detection system also failed to detect fullerene cations. A further I.sub.2 /C.sub.60 cation system using benzene/methanol (50/50, v/v) also failed to detect fullerenes using ESI-MS. Finally, detection of fullerene cations using involatile molecules by gas-phase ion/molecule reactions in a corona discharge mode also failed to detect fullerenes using ESI-MS.
In anion fullerene systems, a reducing agent N,N,N'N'-tetramethyl-p-phenylenediamine (TMPD) in CH.sub.2 Cl.sub.2 and CH.sub.3 OH (60/40, v/v) deoxygenated was also attempted. However, C.sub.70 was not at all detectable and C.sub.60 was only slightly detectable. Finally, the use of NaK amalgam to reduce a mixture of C.sub.60 /C.sub.70 and electrospray with a dimethoxyethane+benzene+methanol (1/6/3, v/v/v) solvent gave uncertain results with many unidentified peaks which were interpreted to include C.sub.60.sup.- and C.sub.70.sup.-. However, very low peaks and resolution made the results uncertain. Due to these results, reduction of C.sub.60 and C.sub.70 would appear to be preferred over oxidation.
Fullerenes
One of the most exciting recent discoveries in chemistry is a new form of elemental carbon, such as C.sub.60 and related forms, also known as buckminster fullerene or buckyball (H. W. Kroto et al. Nature, 518:162 (1985). Until 1985, there were only two known forms of carbon, diamond and graphite. This new form of carbon is a molecule made up of carbon atoms in a shape similar to that of a soccerball. Scientists around the world have begun research into all aspects of buckyballs (T. Bran, Angewate Chem, Int'l Ed,, 31:599 (1992)). The interest and excitement are caused by the unique spherical shape of the molecules as well as its unusual properties. Very recently two new relatives of C.sub.60 (1) nested spheres of buckyballs called buckyonions (S. Iijima, Nature, 305:56 (1991), and (2) hollow fibers called buckytubes (D. Ugarte, Nature, 357:707 (1992)), have been discovered. Buckyballs, may have many possible uses. When buckyballs are mixed with such metals as potassium, they become superconductors (A. F. Hebard et al., Nature, 350:600 (1991)). In addition to their extraordinary mechanical strength, many other unusual properties of these materials have been observed.
In order to take full advantage of buckyballs in materials research, chemists will have to be able to use them as a basis for making other compounds. While the chemistry of carbon compounds is the most diverse in the universe (more than 7,000,000 indexed so far by Chemical Abstracts), buckyball chemical reactivity is poorly understood, partly because of the difficulty in isolating, purifying and identifying buckyball derivatives or "fulleroids".
There are several reasons why buckyballs are difficult to study. First, buckyballs are made up of about 60 or 70 carbon atoms-- and no hydrogen atoms. Thus, nuclear magnetic resonance (NMR) of the hydrogens, the chemist's most useful "photo" of chemical structure, cannot be used. Second, the molecule is very symmetrical but its derivatives are not. For example, one of the simplest derivatives, C.sub.60H.sub.2, has 27 possible isomers. Third, buckyballs are relatively insoluble, and they can be compared to an inert gas in terms of their interactions with solvent molecules. Fourth, while buckyballs are very stable (up to 700.degree. C.) their derivatives are quite fragile, often breaking apart into their parent C.sub.60 or C.sub.70-. A few reactions of buckyballs have been observed and some reviews have appeared (H. Schwarz, Angewante Chem Int'l Ed., 31:293 (1992)).
Fullerenes have been discovered to De mild oxidizing agents, perhaps due to the presence of pyracyclene units which could theoretically capture up to two electrons to give a (4n+2.pi.) electron dianion, or in the form of a lone pair to give a "cyclopentadienide" monoadduct. Due to the high electron affinity of fullerenes, reductions, nucleophilic additions and oxidative additions of low-valent transition metals are considered as the sole avenues of fullerene C.sub.60 functionalization and modification. (Wudl, Acc. Chem. Res. 25:157-161 (1992)).
Accordingly, there is a need to provide MS techniques which allow detection of neutral compounds in solution, as well as for determining reactions and products of reactions, which includes fulleroids.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.